Substantially pure glycopeptide antibotics AC-98-1; AC-98-2; AC-98-3; AC-98-4 AND AC-98-5

ABSTRACT

The invention provides new substantially pure antibiotics designated AC-98-1, AC-98-2, AC-98-3, AC-98-4 and AC-98-5 derived from the microorganism  Streptomyces hygroscopicus.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from copending provisionalapplication Serial No. 60/286,396 filed on Apr. 25, 2001, copendingprovisional application Serial No. 60/286,244 filed on Apr. 25, 2001 andcopending provisional application Serial No. 60/286,249 filed on Apr.25, 2001, the entire disclosures of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to new substantially pure glycopeptideantibiotics, designated AC-98-1, AC-98-2, AC-98-3, AC-98-4 and AC-98-5,or pharmaceutically acceptable salts thereof, to methods for thepreparation and isolation of such antibiotics, to methods of utilizingsuch antibiotics to treat bacterial infections and to pharmaceuticalcompositions containing such antibiotics.

[0004] 2. Description of the Prior Art

[0005] U.S. Pat. No. 3,495,004 discloses a producing organismStreptomyces hygroscopicus NRRL 3085, and production conditions toprepare a complex mixture of antibiotics AC-98. No details of thestructures of the individual antibiotics are disclosed. As described inU.S. Pat. No. 3,495,004, the mixture of AC98 antibiotics could not beseparated into single components by ion exchange (IRC-50), gelfiltration (CM-sephadex), or normal phase chromatography (deactivatedsilica gel).

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 shows the infrared absorption spectrum of AC-98-1

[0007]FIG. 2 shows the infrared absorption spectrum of AC-98-2

[0008]FIG. 3 shows the infrared absorption spectrum of AC-98-3

[0009]FIG. 4 shows the infrared absorption spectrum of AC-98-4

[0010]FIG. 5 shows the infrared absorption spectrum of AC-98-5

[0011]FIG. 6 shows the proton nuclear magnetic resonance spectrum ofAC-98-1

[0012]FIG. 7 shows the proton nuclear magnetic resonance spectrum ofAC-98-2

[0013]FIG. 8 shows the proton nuclear magnetic resonance spectrum ofAC-98-3

[0014]FIG. 9 shows the proton nuclear magnetic resonance spectrum ofAC-98-4

[0015]FIG. 10 shows the proton nuclear magnetic resonance spectrum ofAC-98-5

[0016]FIG. 11 shows the carbon-13 nuclear magnetic resonance spectrum ofAC-98-1

[0017]FIG. 12 shows the carbon-13 nuclear magnetic resonance spectrum ofAC-98-2

[0018]FIG. 13 shows the carbon-13 nuclear magnetic resonance spectrum ofAC-98-3

[0019]FIG. 14 shows the carbon-13 nuclear magnetic resonance spectrum ofAC-98-4

[0020]FIG. 15 shows the carbon-13 nuclear magnetic resonance spectrum ofAC-98-5

SUMMARY OF THE INVENTION

[0021] New substantially pure glycopeptide antibiotics designatedAC-98-1, AC-98-2, AC-98-3, AC-98-4 and AC-98-5 or pharmaceuticallyacceptable salts thereof have now been found.

[0022] The structure of AC-98-1 is:

[0023] The physico chemical characteristics of AC-98-1 are as follows:

[0024] a) Apparent Molecular Formula: C54H78N₁₂O₂₅

[0025] b) Molecular Weight: MS(ESI) [M+2H]²⁺=M/Z 648 (m. w.=1292)HRFABMS calcd. for C₅₄H78N₁₂O₂₅Na=M/Z 1317.5099 HRFABMS observed=M/Z1317.5164 Δ mmu=6.5

[0026] c) Ultraviolet Absorption Spectrum: λ_(max) nm MeOH=280, 273,201;

[0027] d) Infrared Absorption Spectrum: as shown in FIG. 1 (KBr disk):3374, 3277, 1681, 1634,1554, 1510 cm⁻¹;

[0028] e) Proton Magnetic Resonance Spectrum: as shown in FIG. 6 (300MHz, CD₃OD/D₂O 1:1)

[0029] f) Carbon-13 Nuclear Magnetic Resonance Spectrum: as shown inFIG. 11 (75 MHz, CD₃OD/D₂O 1:1), significant peaks are listed below (δfrom TMS);

[0030] 174.4, 173.9, 173.5, 173.5, 173.2, 173.0, 161.9, 161.7, 157.8,144.8, 133.1, 133.0, 131.3, 130.5,129.8, 119.4,104.3,100.8, 83.5, 82.7,76.6, 76.4, 74.9, 74.1, 73.7, 73.5, 73.5, 73.3, 72.2, 72.0, 71.3, 69.5,67.6, 63.8, 63.8, 63.8, 63.5, 62.7, 61.8, 58.3, 58.3, 57.8, 56.8, 56.1,45.0, 44.8, 44.6, 44.1, 38.7, 19.9.

[0031] The structure of AC-98-2 is:

[0032] The physico chemical characteristics of AC-98-2 are as follows:

[0033] a) Apparent Molecular Formula: C₄₂H₅₈N₁₂O₁₅

[0034] b) Molecular Weight: MS(ESI) [M+2H]²⁺=M/Z 486 (m. w.=970) HRFABMScalcd. for C₄₂H₅₈N₁₂O₁₅Na=M/Z 992.4077 HRFABMS observed=M/Z 993.4042 Δmmu=3.5

[0035] c) Ultraviolet Absorption Spectrum: λ_(max) nm MeOH=280, 273,201;

[0036] d) Infrared Absorption Spectrum: as shown in FIG. 2 (KBr disk):3374, 3277, 1681, 1634, 1554, 1510 cm⁻¹;

[0037] e) Proton Magnetic Resonance Spectrum: as shown in FIG. 7 (300MHz, CD₃OD/D₂O 1:1)

[0038] f) Carbon-13 Nuclear Magnetic Resonance Spectrum: as shown inFIG. 12 (75 MHz, CD₃OD/D₂O 1:1), significant peaks are listed below (δfrom TMS);

[0039] 174.3, 173.9, 173.5, 173.6, 173.2, 173.0, 162.0, 161.7, 158.2,144.7, 133.1, 130.0, 131.3,130.5,129.8, 117.8, 83.5, 82.8, 73.7, 72.2,71.9, 71.3, 67.6, 63.8, 63.8, 62.8, 61.8, 58.3, 58.3, 57.8, 56.8, 56.1,45.1, 44.8, 44.5, 44.2, 38.7,19.7.

[0040] The structure of AC-98-3 is:

[0041] The physico chemical characteristics of AC-98-3 are as follows:

[0042] a) Apparent Molecular Formula: C₅₉H₈₆N₁₂O₂₆

[0043] b) Molecular Weight: MS(ESI) [M+2H]²⁺=M/Z 690 (m. w.=1378)HRFABMS calcd. for C₅₉H₈₇N₁₂O₂₆=M/Z 1379.5854 HRFABMS observed=M/Z1379.5861 Δ mmu =0.5

[0044] c) Ultraviolet Absorption Spectrum: λ_(max) nm MeOH=280, 273,201;

[0045] d) Infrared Absorption Spectrum: as shown in FIG. 3 (KBr disk):3374, 3277, 1681, 1634,1554, 1510 cm⁻¹;

[0046] e) Proton Magnetic Resonance Spectrum: as shown in FIG. 8 (300MHz, CD₃OD/D₂O 1:1)

[0047] f) Carbon-13 Nuclear Magnetic Resonance Spectrum: as shown inFIG. 13 (75 MHz, CD₃OD/D₂O 1:1), significant peaks are listed below (δfrom TMS);

[0048] 177.3, 174.4, 173.9, 173.5, 173.5, 173.2, 173.0, 161.9, 161.7,157.8, 144.8, 133.2, 133.1, 131.2, 130.5, 129.8, 119.4, 101.2, 100.8,83.5, 82.8, 76.7, 75.2, 74.8, 73.9, 73.7, 73.6, 71.7, 76.4, 72.2, 72.0,71.3, 69.9, 67.6, 63.9,63.8, 63.8, 63.5, 62.7, 61.8, 58.4,58.3, 57.8,56.8, 56.1, 45.0, 44.8, 44.6, 44.1, 38.7, 19.7, 45.6, 28.2, 24.27,24.24.

[0049] The structure of AC-98-4 is:

[0050] The physico chemical characteristics of AC-98-4 are as follows:

[0051] a) Apparent Molecular Formula: C₅₉H₈₆N₁₂O₂₆

[0052] b) Molecular Weight: MS(ESI) [M+2H]²⁺=M/Z 690 (m. w.=1378)HRFABMS calcd. for C₅₉H₈₇N₁₂O₂₆=M/Z 1379.5854 HRFABMS observed=M/Z1379.5879 Δ mmu=2.5

[0053] c) Ultraviolet Absorption Spectrum: λ_(max) nm MeOH=280, 273,201;

[0054] d) Infrared Absorption Spectrum: as shown in FIG. 4 (KBr disk):3374, 3277, 1681, 1634,1554,1511 cm⁻¹;

[0055] e) Proton Magnetic Resonance Spectrum: as shown in FIG. 9 (300MHz, CD₃OD/D₂O 1:1)

[0056] f) Carbon-13 Nuclear Magnetic Resonance Spectrum: as shown inFIG. 14 (75 MHz, CD₃OD/D₂O 1:1), significant peaks are listed below (δfrom TMS);

[0057] 174.4, 173.9, 173.5, 173.6,173.2, 173.0, 161.8,161.8, 158.0,144.9, 133.2, 133.1, 131.2, 130.5,129.8, 119.5, 104.1, 101.1, 83.7,82.8, 76.6, 76.9, 75.0, 74.2, 73.8, 73.6, 76.4, 71.4, 72.1, 72.1, 71.4,67.0, 67.6, 63.8, 63.8, 63.9, 63.7, 62.7, 61.8, 58.4, 58.4, 57.9, 56.9,56.3, 45.1, 44.9, 44.7, 44.1, 38.8,19.6, 177.6, 45.8, 28.3, 24.3.

[0058] The structure of AC-98-5 is:

[0059] The physico chemical characteristics of AC-98-5 are as follows:

[0060] a) Apparent Molecular Formula: C₅₉H₈₆N₁₂O₂₆

[0061] b) Molecular Weight: MS(ESI) [M+2H]²⁺=M/Z 690 (m. w.=1378)HRFABMS calcd. for C₅₉H₈₆N₁₂O₂₆Na=M/Z 1401.5674 HRFABMS observed=M/Z1401.5693 Δ mmu=1.9

[0062] c) Ultraviolet Absorption Spectrum: λ_(max) nm MeOH=280, 273,201;

[0063] d) Infrared Absorption Spectrum: as shown in FIG. 5 (KBr disk):3374, 3277, 1681, 1634,1554, 1510 cm⁻¹;

[0064] e) Proton Magnetic Resonance Spectrum: as shown in FIG. 10 (300MHz, CD₃OD/D₂O 1:1)

[0065] f) Carbon-13 Nuclear Magnetic Resonance Spectrum: as shown inFIG. 15 (75 MHz, CD₃OD/D₂O 1:1), significant peaks are listed below (δfrom TMS);

[0066] 177.1, 174.7, 173.9, 173.6,173.7,173.3, 173.1, 162.1, 161.9,157.9,144.9, 133.1, 133.1, 131.2, 130.5, 129.7, 119.4, 104.3, 101.0,83.9, 82.9, 76.7, 74.7, 75.0, 74.2, 73.9, 73.6, 71.8, 73.7, 72.3, 72.1,71.6, 72.0, 67.7, 63.8, 63.8, 63.9, 64.3, 62.6, 61.9, 58.5, 58.6, 58.0,57.0, 56.5, 45.1, 45.0, 44.8, 44.0, 38.8, 19.5, 45.9, 28.3, 24.36,24.33.

[0067] In particular the structures of substantially pure AC-98-1,AC-98-2, AC-98-3, AC-98-4 and AC-98-5 are:

[0068] This invention provides a method of preparing, separating andisolating substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4, and AC-98-5 from a recovered complex mixture.

[0069] This invention further provides a method for preparingsubstantially pure glycopeptide antibiotic AC-98-1 comprising the stepsof:

[0070] a. cultivating a suitable producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-1;

[0071] b. recovering said mixture of AC-98 antibiotics containingAC-98-1; and

[0072] c. separating and isolating substantially pure AC-98-1 as thetrifluoroacetic acid salt by reverse phase high pressure liquidchromatography with a mobile phase gradient of about 11% to about 25%acetonitrile in water containing about 0.02% trifluoroacetic acid.

[0073] Also preferred is a mobile phase gradient of about 40% to about60% methanol in water containing about 0.02% trifluoroacetic acid.

[0074] This invention further provides a method for preparingsubstantially pure glycopeptide antibiotic AC-98-2 comprising the stepsof:

[0075] a. cultivating a suitable a producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-2;

[0076] b. recovering said mixture of AC-98 antibiotics containingAC-98-2; and

[0077] c. separating and isolating substantially pure AC-98-2 as thetrifluoroacetic acid salt by reverse phase high pressure liquidchromatography with a mobile phase gradient of about 11% to about 25%acetonitrile in water containing about 0.02% trifluoroacetic acid.

[0078] Also preferred is a mobile phase gradient of about 40% to about60% methanol in water containing about 0.02% trifluoroacetic acid.

[0079] This invention further provides a method for preparingsubstantially pure glycopeptide antibiotic AC-98-3 comprising the stepsof:

[0080] a. cultivating a suitable producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-3;

[0081] b. recovering said mixture of AC-98 antibiotics containingAC-98-3; and

[0082] c. separating and isolating substantially pure AC-98-3 as thetrifluoroacetic acid salt by reverse phase high pressure liquidchromatography with a mobile phase gradient of about 11% to about 25%acetonitrile in water containing about 0.02% trifluoroacetic acid.

[0083] Also preferred is a mobile phase gradient of about 40% to about60% methanol in water containing about 0.02% trifluoroacetic acid.

[0084] This invention further provides a method for preparingsubstantially pure glycopeptide antibiotic AC-98-4 comprising the stepsof:

[0085] a. cultivating a suitable producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-4;

[0086] b. recovering said mixture of AC-98 antibiotics containingAC-98-4; and

[0087] c. separating and isolating substantially pure AC-98-4 as thetrifluoroacetic acid salt by reverse phase high pressure liquidchromatography with a mobile phase gradient of about 11% to about 25%acetonitrile in water containing about 0.02% trifluoroacetic acid.

[0088] Also preferred is a mobile phase gradient of about 40% to about60% methanol in water containing about 0.02% trifluoroacetic acid.

[0089] This invention further provides a method for preparingsubstantially pure glycopeptide antibiotic AC-98-5 comprising the stepsof:

[0090] a. cultivating a suitable producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-5;

[0091] b. recovering said mixture of AC-98 antibiotics containingAC-98-5; and

[0092] c. separating and isolating substantially pure AC-98-5 as thetrifluoroacetic acid salt by reverse phase high pressure liquidchromatography with a mobile phase gradient of about 11% to about 25%acetonitrile in water containing about 0.02% trifluoroacetic acid.

[0093] Also preferred is a mobile phase gradient of about 40% to about60% methanol in water containing about 0.02% trifluoroacetic acid.

[0094] It is understood that this invention encompasses all crystallineforms of substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4 and AC-98-5. Further, substantially pure antibioticsAC-98-1, AC-98-2, AC-98-3, AC-98-4 and AC-98-5 may be obtained aspharmaceutically acceptable salts which are those derived from suchorganic and inorganic acids as: acetic, trifluoroacetic, lactic, citric,tartaric, formate, succinic, maleic, malonic, gluconic, hydrochloric,hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, andsimilarly known acceptable acids. The pharmaceutically acceptable saltsof compounds of the invention are prepared using conventionalprocedures.

[0095] Substantially pure compounds of the invention have centers ofasymmetry. The substantially pure compounds may, therefore, exist in atleast two and often more stereoisomeric forms. The present inventionencompasses all stereoisomers of the substantially pure compoundswhether free from other stereoisomers or admixed with otherstereoisomers in any proportion and thus includes, for instance, racemicmixtures of enantiomers as well as the diastereomeric mixtures ofisomers. The absolute configuration of any substantially pure compoundmay be determined by any suitable method including conventional X-raycrystallography.

[0096] The present invention accordingly provides a pharmaceuticalcomposition which comprises a substantially pure glycopeptide antibioticAC-98-1, AC-98-2, AC-98-3, AC-98-4 or AC-98-5 or a mixture thereof incombination or association with a pharmaceutically acceptable carrier.In particular, the present invention provides a pharmaceuticalcomposition which comprises an effective amount of substantially pureAC-98-1, AC-98-2, AC-98-3, AC-98-4 or AC-98-5 or a mixture thereof and apharmaceutically acceptable carrier.

[0097] The present invention also provides methods which may be used intreating bacterial infections in warm blooded animals which compriseadministering to said animals an antibacterially effective amount of asubstantially pure AC-98-1, AC-98-2, AC-98-3, AC-98-4 or AC-98-5 or amixture thereof. Warm blooded animals includes humans.

DETAILED DESCRIPTION OF THE INVENTION

[0098] New substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4 and AC-98-5 are obtained from a complex AC-98antibiotic mixture which is produced by aerobic fermentation of theculture (Streptomyces hygroscopicus) NRRL 3085 using the conditions asdescribed in U.S. Pat. No. 3,495,004. This culture is maintained in theculture collection of Wyeth-Ayerst Research, Pearl River, N.Y. asculture number AC-98. A viable culture of this microorganism has beendeposited, with the ARS Culture Collection, Fermentation Laboratory,Northern Regional Research Center, U.S. Department of Agriculture, 1815North University Street, Peoria, Ill. 61604 and has been added to itspermanent collection and assigned the strain designation NRRL 3085.Another suitable producing strain of Streptomyces hygroscopicus is NRRL4600 (NRRL30439) Cultivating of (Streptomyces hygroscopicus) NRRL 3085and recovering mixtures of AC-98 antibiotics following silica gel andweakly acidic cation exchange resin chromatography is described in U.S.Pat. No. 3,495,004, incorporated herein by reference. The mixtures ofAC-98 antibiotics from eleven like cultivations are combined, dissolvedin water and butanol and evaporated to a residue which is further heatedin methanol, centrifuged and collected as a AC-98 antibiotic mixturefollowing washing with methanol and acetone. Separating the AC-98antibiotic mixture into substantially pure AC-98-1, AC-98-2, AC-98-3,AC-98-4 or AC-98-5 is described in the present application.

[0099] Experimental efforts showed that the AC-98 mixture could not beeffectively separated into substantially pure AC-98-1, AC-98-2, AC-98-3,AC-98-4 and AC-98-5 using reverse phase HPLC on C-18 columns whichincluded Dynamax and Phenomenex C-18 columns (60A pore size, 5 or 8 μmparticle size) using acetonitrile/water or methanol/water (both withadjusted pHs) as the solvents. Surprisingly, however, the separationresolution was accomplished by using YMC ODS-A columns (120A pore size,5 or 10 μm particle size). As determined experimentally, the HPLC isperformed with C18 reverse phase columns (YMC ODS-A, 120A pore size)using mixtures of acetonitrile or methanol in water containing smallamounts of trifluoroacetic acid to control the acidity in the range ofpH 3.5 and 5.5. The purification of the substantially pure antibioticsAC-98-1, AC-98-2, AC-98-3, AC-98-4, and AC-98-5 from the AC-98antibiotic mixture is finally achieved by dissolving the AC-98 mixturein water or water/methanol mixture and subjecting the resulting solutionto reverse phase HPLC.

[0100] Separating the substantially pure AC-98-1, AC-98-2, AC-98-3,AC-98-4 or AC-98-5 each from the others is accomplished using reversephase HPLC on a C18 column (YMC ODS-A, 8 μm particle size, 20×250 mm)using a mobile phase consisting of a gradient from about 11% to about25% by volume of acetonitrile in water containing about 0.02%trifluoroacetic. The flow rate of the mobile phase is maintainedconstant at 9 ml/minute and the effluent monitored by UV absorbance at226 nm. Substantially pure AC-98-5 is further separated using reversephase HPLC and a mobile phase gradient of about 40% to about 60%methanol in water containing about 0.02% trifluoroacetic acid.

[0101] Substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4 and AC-98-5 of this invention are defined as having, apurity of at least 85% when separated each from the others, asdetermined by high pressure liquid chromatography(HPLC). Preferably,substantially pure AC-98-1 is obtained with a purity of at least 92%,substantially pure AC-98-2 is obtained with a purity of at least 94%,substantially pure AC-98-3 is obtained with a purity of at least 89%,substantially pure AC-98-4 is obtained with a purity of at least 91% andsubstantially pure AC-98-5 is obtained with a purity of at least 89%.

[0102] The substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4, and AC-98-5 are isolated, purified and characterizedfrom the AC-98 antibiotic mixture by dissolving the mixture in water ora water/methanol mixture and subjecting the resulting solution toreverse phase chromatography. Typically the chromatography is performedwith C18 reverse phase media using mixtures of acetonitrile or methanolin water containing small amounts of organic acids, such astrifluoroacetic acid to control the acidity in the range of pH 3.5 and5.5.

[0103] Cultivation of suitable producing strains of Streptomyceshygroscopicus may be carried out in a wide variety of suitable liquidculture media. Media which are useful for the production of AC-98glycopeptide antibiotics include an assimilable source of carbon, suchas dextrin, dextrose, sucrose, molasses, starch, glycerol, etc; anassimilable source of nitrogen such as protein, protein hydrolysate,polypeptides, amino acids, corn steep liquor, etc; and inorganic anionsand cations, such as potassium, sodium, ammonium, calcium, sulfate,carbonate, phosphate, chloride, etc. Trace elements such as zinc,cobalt, iron, boron, molybdenum, copper, etc., are supplied asimpurities of other constituents of the media. Aerobic conditionsinclude aeration in tanks and bottles supplied by forcing sterile airthrough or onto the surface of the fermenting medium. Further agitationin tanks is provided by a mechanical impeller. An antifoam agent such aspolypropylene glycol may be added as needed. In general, cultivating asuitable producing strain of Streptomyces hygroscopicus in a suitableculture medium is continued for about 24 to about 240 hours to produce amixture of AC-98 antibiotics containing AC-98-1, AC-98-2, AC-98-3,AC-98-4 and AC-98-5. In particular, suitable liquid culture media arelisted in Table A.

[0104] Culture Preservation

[0105] Cultures may be preserved as frozen whole cells (frozenvegetative mycelia, FVM) at −70° C. Glycerol may be added to cells grownfor 24-48 hours in TSBG (Tryptic soy broth [Difco] supplemented with 20g/L glucose) to a final concentration of 20%. The suspension may then bealiquoted to cryovials and frozen.

Inoculum Development and Growth of Organisms for Preparation of FrozenStock Cultures

[0106] Fermentations may be inoculated from “seed” stage cells grown inTSBG medium. Inoculum cells may be grown in various configurationsdepending on the fermentation scale, such as in test tubes containing 10ml medium, shake-flasks containing 25, 50, 250 or 500 ml medium or in10-liter fermentor. Primary seeds (tube, flask) may be inoculated fromFVM at 0.2-5.0% and incubated on rotary shaker at 30° C. for 24-48hours. Primary seeds may be used for inoculation of shake-flask andten-liter fermentors. A second stage, ten-liter TSBG seed fermentor maybe used for inoculation of 300-liter fermentations. Seed fermentors maybe operated at 30° C., 500 rpm with 1 vvm air for 24-48 hours. TABLE AComposition of fermentation media BPM17st BPM17st BPM27 Component BPM17BPM17stat atgal atman BPM27 -man Pharmame 10 g/L 20 g/L 20 g/L 20 g/L 20g/L 20 g/L dia (Traders) Glucose 40 g/L 60 g/L 60 g/L 60 g/L 60 g/L 60g/L Galactose — — 20 g/L — — — Mannose — — —  2 g/L  2 g/L — CaCO₃  5g/L  5 g/L  5 g/L  5 g/L — — (Mississippi Lime) CaCO₃ — — — — 15 g/L 15g/L (Gamaco)

[0107] Shake-flask fermentations may be performed at 30° C. on agyro-rotary shaker operating at 250 rpm (2″ stroke) for from 3-5 days.Ten-liter fermentations may be performed at 30° C. for 3-5 days at 30°C., at 400-800 rpm with1 vvm airflow. Fermentation at 300 liters may beperformed similarly with agitation at 170-200 rpm. A polypropyleneglycol antifoam, such as Macol P2000 may be added to fermentor medium at0.2-2.0%. Three hundred-liter fermentations with medium BPM17statgalemploy galactose at 8 g/L rather than the smaller scale concentration of20 g/L.

LC/MS Analysis of Glycopeptide Antibiotics

[0108] The molecular weights of new substantially pure AC-98glycopeptide antibiotics may be determined using a Hewlett-PackardAPI-electrospray LC/MS system with an HP 5989B Mass Spectrometer, HP59987A API-Electrospray, HP 1090 series 11 HPLC and HP ChemStation datasystem with HP G1047A LC/MS software and UV detection at 226 nm. The MSelectrospray may be performed in positive mode with a scan range of400˜1700 m/z.

Optional Procedure for Isolation of a Mixture of AC-98 GlycopeptideAntibiotics

[0109] Optionally, glycopeptide antibiotics of the AC-98 complex may beisolated from fermentations by passing supernatant through a columncontaining methacrylate resin XAD-7. The mixture of glycopeptideantibiotics of the AC-98 complex retained on the column may then beeluted by a mixed solvent of acetonitrile in water containing a smallamount of acid. After solvent is evaporated under reduce pressure, theindividual compounds may be purified by reverse phase chromatography.Typically, the chromatography is performed with C18 reverse phase mediausing mixtures of acetonitrile or methanol in water with small amount ofacid for pH control, the preferred acid being trifluoroacetic acid.

[0110] Substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4, and AC-98-5 derive their utility from theirantibacterial activity. In particular the substantially pure antibioticsAC-98-1, AC-98-2, AC-98-3, AC-98-4 and AC-98-5 are active againstmethicillin-susceptible and methicillin-resistant strains ofstaphylococci, against penicillin-susceptible and penicillin-resistantstreptococci, and against vancomycin-susceptible andvancomycin-resistant enterococci. Further, for example, these compoundsmay be used in the suppression of bacterial infections, as a topicalantibacterial agent and as a general disinfectant for laboratories.

[0111] In therapeutic use, the substantially pure glycopeptideantibiotics AC-98-1, AC-98-2, AC-98-3, AC-98-4, and AC-98-5 of thisinvention may be administered in the form of conventional pharmaceuticalcompositions appropriate for the intended use. Such compositions may beformulated so as to be suitable for oral, parenteral or topicaladministration. The substantially pure glycopeptide antibiotics AC-98-1,AC-98-2, AC-98-3, AC-98-4, and AC-98-5 may be combined in admixture witha nontoxic pharmaceutical carrier, which carrier may take a variety offorms, depending on the form of preparation desired for administration,ie. oral, parenteral, or topical.

[0112] When the substantially pure glycopeptide antibiotics AC-98-1,AC-98-2, AC-98-3, AC-98-4, and AC-98-5 are employed for the aboveutility, they can be combined with one or more pharmaceuticallyacceptable carriers, for example, solvents, diluents and the like, andmay be administered orally in such forms as tablets, capsules,dispersible powders, granules, or suspensions containing, for example,from about 0.05 to 5% of suspending agent, syrups containing, forexample from about 10 to 50% of sugar, and elixirs containing, forexample from about 20 to 50% ethanol, and the like, or parenterally inthe form of sterile injectable solutions or suspensions containing fromabout 0.05 to 5% suspending agent in an isotonic medium. Suchpharmaceutical preparations may contain, for example, from about 0.05 upto about 90% of the substantially pure glycopeptide antibiotics AC-98-1,AC-98-2, AC-98-3, AC-98-4, and AC-98-5 in combination with the carrier,more usually between about 5% and 60% by weight.

[0113] An antibacterially effective amount of substantially pureglycopeptide antibiotics AC-98-1, AC-98-2, AC-98-3, AC-98-4 or AC-98-5from about 0.5 mg/kg of body weight to about 200.0 mg/kg of body weightshould be administered one to five times per day via any topical routesof administration including but not limited to oral, parenteral(including subcutaneous, intravenous, intramuscular, intrasternalinjection or infusion techniques), by inhalation spray, or rectally, indosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. It will beunderstood, however, that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sec, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the host undergoing therapy.

[0114] Additionally, the antibacterially effective amount of thesubstantially pure glycopeptide antibiotics AC-98-1, AC-98-2, AC-98-3,AC-98-4 or AC-98-5 may be administered at a dosage and frequency withoutinducing side effects commonly experienced with conventional antibiotictherapy which could include hypersensitivity, neuromuscular blockade,vertigo, photosensitivity, discoloration of teeth, hematologic changes,gastrointestinal disturbances, ototoxicity, and renal, hepatic, orcardiac impairment. Further the frequency and duration of dosage may bemonitored to substantially limit harmful effects to normal tissuescaused by administration at or above the antibacterially effectiveamount of the substantially pure glycopeptide antibiotics AC-98-1,AC-98-2, AC-98-3, AC-98-4 or AC-98-5.

[0115] These active compounds may be administered orally as well as byintravenous, intramuscular, or subcutaneous routes. Solid carriersinclude starch, lactose, dicalcium phosphate, microcrystallinecellulose, sucrose and kaolin, while liquid carriers include sterilewater, polyethylene glycols, non-ionic surfactants and edible oils suchas corn, peanut and sesame oils, as are appropriate to the nature of theactive ingredient and the particular form of administration desired.Adjuvants customarily employed in the preparation of pharmaceuticalcompositions may be advantageously included, such as flavoring agents,coloring agents, preserving agents, and antioxidants, for example,vitamin E, ascorbic acid, BHT and BHA.

[0116] The preferred pharmaceutical compositions from the stand-point ofease of preparation and administration are solid compositions,particularly tablets and hard-filled or liquid-filled capsules. Oraladministration of the compound is preferred.

[0117] These substantially pure glycopeptide antibiotics AC-98-1,AC-98-2, AC-98-3, AC-98-4, and AC-98-5 may also be administeredparenterally or intraperitoneally. Solutions or suspensions of theseactive compounds as a free base or pharmacologically acceptable salt canbe prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

[0118] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microrganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oil.

[0119] The invention will be further described in conjunction with thefollowing non-limiting examples.

EXAMPLE 1 Preparation of a Mixture of AC-98 Antibiotics

[0120] A mixture of AC-98 antibiotics is prepared following thefermentation description in Example 3 of U.S. Pat. No. 3,495,004 whereinthe antibiotic containing eluate is concentrated and further purified bypassing through a column of deactivated silica gel followed by a weaklyacidic cation exchange resin, IRC-50. Elution of the resin by acidicaqueous solution, followed by evaporation in vacuo affords a mixture ofglycopeptide antibiotics AC-98. The mixture of antibiotics from elevenindividually isolated fermentation batches are combined to afford 23.626g of a mixture of AC-98 antibiotics which is dissolved in 500 ml ofwater containing a minimum volume of butyl alcohol and filtered. Thefiltrate is evaporated in vacuo with the further addition of butylalcohol and continuous evaporation while removing excess water to afforda residue. To the residue is added 100 ml of methyl alcohol followed byheating to the boil and centrifuging to a solid. The solid is washedwith methyl alcohol then acetone followed by centrifuging to afford19.841 g of a mixture of AC-98 antibiotics as a solid after rewashingwith acetone and drying.

[0121] The mixture of AC-98 antibiotics is analyzed by HPLC to containmainly five components, designated as AC-98-1 (17%), AC-98-2 (19%),AC-98-3 (15%), AC-98-4 (29%), and AC-98-5 (4%). The relative quantity ofeach antibiotic is calculated based on the integration area at 226 nm.Referring to the remaining 16% of the mixture of AC-98 antibiotics,approximately 15% belong to components which have a different UV spectraand less than 1% further minor components of AC-98 antibiotics. Theanalysis is performed by HPLC on a Cl 8 column (YMC ODS-A, 5 μm particlesize, 4.6×150 mm). The mobile phase, monitored by UV detector at 226 nm,is a gradient of acetonitrile (10% to 50%) in water containing 0.01%trifluoroacetic acid over 22 minutes at a flow rate of 1 ml per minute.The relative quantity of each glycopeptide antibiotic is calculatedbased on the integration area at 226 nm.

EXAMPLE 2 Substantially Pure Glycopeptide Antibiotics AC-98-1, AC-98-2,AC-98-3, and AC-98-4 From a Mixture of AC-98 Antibiotics

[0122] A mixture of AC-98 antibiotics(180 mg) from Example 1 isdissolved in water (1 ml) and subjected to reverse phase HPLC on a C18column (YMC ODS-A, 8 μm particle size, 20×250 mm). The mobile phaseconsisting of a gradient from 11% to 25% by volume of acetonitrile inwater containing 0.02% trifluoroacetic acid over 45 minutes. The flowrate of the mobile phase is maintained constant at 9 ml/minute and theeffluent monitored by UV absorbance at 226 nm. Individually collectedfractions having the same retention times by HPLC are pooled and uponevaporation infrared, proton nuclear magnetic resonance, and carbon 13magnetic resonance spectra recorded. The substantially pure glycopeptideantibiotics AC-98-1, AC-98-2, AC-98-3, AC-98-4 and a mixture astrifluoroacetate salts are isolated and listed in Table 1. TABLE 1WEIGHT COMPONENT RETENTION TIME COLLECTED* Substantially Pure AC-98-1 20MINUTES 35 mg Substantially Pure AC-98-2 28 MINUTES 29 mg SubstantiallyPure AC-98-3 32 MINUTES 25 mg Substantially Pure AC-98-4 37 MINUTES 64mg Mixture 43 MINUTES 13 mg

EXAMPLE 3 Isolation of Substantially Pure Glycopeptide AntibioticAC-98-5 Trifluoroacetate

[0123] The mixture from Table 1 (13 mg) eluted at 43 minutes isdissolved in water (200 μl) and subjected to reverse phase HPLC on a C18column (YMC ODS-A, 8 μm particle size, 20×250 mm). The mobile phase is agradient of methanol (40% to 60%) in water containing 0.02%trifluoroacetic acid over 40 minutes at a flow rate of 9 ml/minute. Themajor peak centers at 29 minutes, as monitored by UV detector at 226 nm,and upon evaporation infrared, proton nuclear magnetic resonance andcarbon 13 magnetic resonance spectra are recorded. The substantiallypure AC-98-5 (5 mg) is isolated as a trifluoroacetate salt.

[0124] The purities of the five substantially pure glycopeptideantibiotics separated by methods described in Examples 1-3, aredetermined by HPLC analyses (226 nm) to be:

[0125] AC98-1 - - - 92%

[0126] AC98-2 - - - 94%

[0127] AC98-3 - - - 89%

[0128] AC98-4 - - - 91%

[0129] AC98-5 - - - 89%

[0130] The substantially pure glycopeptide antibiotics AC-98-1, AC-98-2,AC-98-3, AC-98-4, and AC-98-5 are tested in the following standardpharmacological test procedures.

Biological Activity

[0131] The in vitro antibacterial activity of substantially pureglycopeptide antibiotics AC-98-1, AC-98-2, AC-98-3, AC-98-4, and AC-98-5from Examples 2 and 3 is determined against a spectrum of bacteria by astandard agar dilution method. Mueller-Hinton agar containing 5% sheepblood and two-fold decreasing concentrations of substantially pureglycopeptide antibiotics AC-98-1, AC-98-2, AC-98-3, AC-98-4, and AC-98-5from Examples 2 and 3 are poured into petri dishes. The agar surfacesare inoculated with 1 to 5×10⁴ colony forming units of bacteria by meansof a Steers replicating device. The lowest concentration of antibioticthat inhibited the growth of a bacterial strain after 18 hoursincubation is recorded as the minimal inhibitory concentration for thatstrain. The results are given in Table II. TABLE II In vitroantibacterial activity of substantially pure antibiotics AC-98-1,AC-98-2, AC-98-3, AC-98-4, and AC-98-5 from Examples 2 and 3 MIC (mg/mL)Organism AC-98-1 AC-98-2 AC-98-3 AC-98-4 AC-98-5 Staphylococcus aureus(NEMC-89-4) >128 64 8 8 4 Staphylococcus aureus (ID-2371) >128 128 8 8 4Staphylococcus aureus (ID-2727) >128 64 8 8 4 Staphylococcus aureus(SMITH) 128 64 8 8 4 Staphylococcus aureus (ID-3105) 128 64 8 8 4Staphylococcus aureus (ID-4379) 128 64 8 4 4 Staphylococcus aureus (ATCC29213) 128 64 8 8 4 Staphylococcus hemolyticus (ID-4061) 128 32 8 4 4Staph Coagulase Neg (ID-3135) 128 32 8 4 2 Staph Coagulase Neg (ID-3276)128 64 4 4 2 Staph Coagulase Neg (ID-3120) 128 64 8 8 2 Staph CoagulaseNeg (ID-3941) 64 32 4 2 2 Staph Coagulase Neg (4615) >128 128 8 8 4Enterococcus faecalis (ID-4168) >128 >128 64 64 16 Enterococcus faecalis(ID-1829) >128 128 64 64 32 Enterococcus faecalis (ID-2131) >128 >128128 64 32 Enterococcus faecalis (12201) >128 >128 64 32 16 Enterococcusfaecalis (ATCC 29212) >128 >128 64 64 16 Enterococcus faecium(12202) >128 128 64 64 32 Enterococcus faecium (ID-3301) >128 >128 64 6416 Enterococcus faecium (ID-4133) 128 32 16 8 4 Enterococcus faecium(ID-3953) >128 128 64 64 32 Streptococcus pyogenes (ID-3187) >32 64 8 82 Streptococcus pneumoniae (ID-4444) >32 >64 8 4 2 Streptococcuspneumoniae (GC1889) 4 4 Pseudomonas aeruginosa >128 >128 >128 >128 >128(ATCC 27853) Morganella morganii (VGH 84-11) >128 >128 >128 >128 >128Escherichia coli (J2175) >128 64 >128 128 64 Escherichia coli (J2445) 6432 4 4 1 Escherichia coli (ATCC 25922) >128 64 >128 >128 128 Bacillussubtilis (Bacto) >128 >128 16 16 8 Micrococcus luteus (ATCC 9341) 64 162 2 1

[0132] The in vivo antibacterial activity of substantially pureglycopeptides AC-98-1, AC-98-2, AC-98-3, AC-98-4, and AC-98-5 isestablished by infecting female CD-1 mice from Charles RiverLaboratories, weighing 20+/−2 g each, intraperitoneally with 6.5×10⁵CFU/0.5 ml of broth of Staphylococcus aureus Smith. The mice are treatedintravenously, 30 minutes before infection with the indicated dose ofthe test compound in 0.2 ml of water. The results of this test are givenin Table III. TABLE III In vivo antibacterial activity of substantiallypure glycopeptide antibiotics AC-98-1, AC-98-2, AC-98-3, AC-98-4, andAC-98-5 from Examples 2 and 3 ED₅₀ (iv, mg/kg) Compound Staphylococcusaureus AC-98-1 20 AC-98-2 >32 AC-98-3 3.8 AC-98-4 2.6 AC-98-5 0.6

We claim:
 1. A substantially pure compound having the structure

or pharmaceutically acceptable salts, thereof.
 2. A substantially purecompound according to claim 1 and having the structure


3. A method for treating bacterial infections in warm blooded animalswhich comprises providing to said animals an antibacterially effectiveamount of a compound according to claim
 2. 4. A pharmaceuticalcomposition which comprises a compound according to claim 2 inassociation with a pharmaceutically acceptable carrier.
 5. Asubstantially pure compound having the structure

or pharmaceutically acceptable salts thereof.
 6. A substantially purecompound according to claim 5 having the structure


7. A method for treating bacterial infections in warm blooded animalswhich comprises providing to said animals an antibacterially effectiveamount of a compound according to claim
 6. 8. A pharmaceuticalcomposition which comprises a compound according to claim 6 inassociation with a pharmaceutically acceptable carrier.
 9. Asubstantially pure compound having the structure

or pharmaceutically acceptable salts thereof.
 10. A substantially purecompound according to claim 9

or pharmaceutically acceptable salts thereof.
 11. A method for treatingbacterial infections in warm blooded animals which comprises providingto said animals an antibacterially effective amount of a compoundaccording to claim
 10. 12. A pharmaceutical composition which comprisesa compound according to claim 10 in association with a pharmaceuticallyacceptable carrier.
 13. A substantially pure compound having thestructure

or a pharmaceutically acceptable salt thereof.
 14. A substantially purecompound according to claim 13 having the structure


15. A method for treating bacterial infections in warm blooded animalswhich comprises providing to said animals an antibacterially effectiveamount of a compound according to claim
 14. 16. A pharmaceuticalcomposition which comprises a compound according to claim 14 inassociation with a pharmaceutically acceptable carrier.
 17. Asubstantially pure compound having the structure

or a pharmaceutically acceptable salt thereof.
 18. A substantially purecompound according to claim 17 having the structure

or a pharmaceutically acceptable salt thereof.
 19. A method for treatingbacterial infections in warm blooded animals which comprises providingto said animals an antibacterially effective amount of a compoundaccording to claim
 18. 20. A pharmaceutical composition which comprisesa compound according to claim 18 in association with a pharmaceuticallyacceptable carrier.
 21. A method for preparing substantially pureglycopeptide antibiotic AC-98-1 comprising the steps of: a. cultivatinga suitable producing strain of Streptomyces hygroscopicus in a suitableculture medium under aerobic conditions to produce a mixture of AC-98antibiotics containing AC-98-1; b. recovering said mixture of AC-98antibiotics containing AC-98-1;and c. separating and isolatingsubstantially pure AC-98-1 as the trifluoroacetic acid salt by reversephase high pressure liquid chromatography with a mobile phase gradientof about 11% to about 25% acetonitrile in water containing about 0.02%trifluoroacetic acid.
 22. The method according to claim 21 where themobile phase is a gradient of about 40% to about 60% methanol in watercontaining about 0.02% trifluoroacetic acid.
 23. A method for preparingsubstantially pure glycopeptide antibiotic AC-98-2 comprising the stepsof: a. cultivating a suitable producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-2; b. recoveringsaid mixture of AC-98 antibiotics containing AC-98-2;and c. separatingand isolating substantially pure AC-98-2 as the trifluoroacetic acidsalt by reverse phase high pressure liquid chromatography with a mobilephase gradient of about 11% to about 25% acetonitrile in watercontaining about 0.02% trifluoroacetic acid.
 24. The method according toclaim 23 where the mobile phase is a gradient of about 40% to about 60%methanol in water containing about 0.02% trifluoroacetic acid.
 25. Amethod for preparing substantially pure glycopeptide antibiotic AC-98-3comprising the steps of: a. cultivating a suitable producing strain ofStreptomyces hygroscopicus in a suitable culture medium under aerobicconditions to produce a mixture of AC-98 antibiotics containing AC-98-3;b. recovering said mixture of AC-98 antibiotics containing AC-98-3;andc. separating and isolating substantially pure AC-98-3 as thetrifluoroacetic acid salt by reverse phase high pressure liquidchromatography with a mobile phase gradient of about 11% to about 25%acetonitrile in water containing about 0.02% trifluoroacetic acid. 26.The method according to claim 25 where the mobile phase is a gradient ofabout 40% to about 60% methanol in water containing about 0.02%trifluoroacetic acid.
 27. A method for preparing substantially pureglycopeptide antibiotic AC-98-4 comprising the steps of: a. cultivatinga suitable producing strain of Streptomyces hygroscopicus in a suitableculture medium under aerobic conditions to produce a mixture of AC-98antibiotics containing AC-98-4; b. recovering said mixture of AC-98antibiotics containing AC-98-4;and c. separating and isolatingsubstantially pure AC-98-4 as the trifluoroacetic acid salt by reversephase high pressure liquid chromatography with a mobile phase gradientof about 11% to about 25% acetonitrile in water containing about 0.02%trifluoroacetic acid.
 28. The method according to claim 27 where themobile phase is a gradient of about 40% to about 60% methanol in watercontaining about 0.02% trifluoroacetic acid.
 29. A method for preparingsubstantially pure glycopeptide antibiotic AC-98-5 comprising the stepsof: a. cultivating a suitable producing strain of Streptomyceshygroscopicus in a suitable culture medium under aerobic conditions toproduce a mixture of AC-98 antibiotics containing AC-98-5; b. recoveringsaid mixture of AC-98 antibiotics containing AC-98-5;and c. separatingand isolating substantially pure AC-98-5 as the trifluoroacetic acidsalt by reverse phase high pressure liquid chromatography with a mobilephase gradient of about 11% to about 25% acetonitrile in watercontaining about 0.02% trifluoroacetic acid.
 30. The method according toclaim 29 where the mobile phase is a gradient of about 40% to about 60%methanol in water containing about 0.02% trifluoroacetic acid.